Malaria Transmittance
Malaria is a mosquito-borne disease caused by a parasite. At least four species of malaria parasites can infect humans under natural conditions: Plasmodium falciparum, P. vivax, P. ovale and P. malariae. The first two species cause the most infections worldwide. P. vivax and P. ovale have dormant liver stage parasites (hypnozoites) that can reactivate (or “relapse”) and cause malaria several months or years after the infecting mosquito bite; consequently, these species can be difficult to detect in infected individuals.
In nature, malaria parasites spread by infecting successively two types of hosts: humans and female Anopheles mosquitoes. In humans, the parasites grow and multiply first in the liver cells and then in the red blood cells. In the blood, successive broods of parasites grow inside the red cells and destroy them, releasing daughter parasites (merozoites) that continue the cycle by invading other red cells.
The blood stage parasites cause the symptoms of malaria. When certain forms of blood stage parasites, gametocytes, are picked up by a female Anopheles mosquito during a blood meal, they start another, different cycle of growth and multiplication in the mosquito. After 10-18 days, the parasites are found as sporozoites in the mosquito's salivary glands. When the Anopheles mosquito takes a blood meal from another human, the sporozoites are injected with the mosquito's saliva and start another human infection when they parasitize the liver cells (Wyler, 1992).
Malaria Symptoms and Disease
Infection with malaria parasites can result in a wide variety of symptoms, ranging from absent or very mild symptoms to severe disease and even death. Malaria disease can be categorized as uncomplicated or (complicated) severe. In general, malaria is curable if diagnosed and treated promptly. Following the infective mosquito bite there is an incubation period before the first symptoms appear. The incubation period usually varies from 7 to 30 days. The shorter periods are observed most frequently with P. falciparum and the longer with P. vivax. In fact, P. vivax can have extended incubation periods, over 450 days (Lee et al., 1998).
Diagnosis
Malaria must be recognized promptly in order to treat the patient in time and to prevent further spread of infection in the community. Because of the long incubation period for P. vivax, diagnosis can be difficult by traditional blood smear methods, delaying treatment. Delay in diagnosis and treatment is a leading cause of death in malaria patients. Malaria can be suspected based on a patient's symptoms and physical findings at examination. However, for a definitive diagnosis, laboratory tests must demonstrate presence of the malaria parasites. The present diagnostic “gold standard” for malaria depends on the demonstration of parasites on a blood smear examined under a microscope.
Detection of Plasmodium Antibodies
Antibodies to asexual malaria parasites (i.e., merozoites) appear within days to weeks after the parasites invade erythrocytes and can persist for months or even years (Vinetz et al., 1998). Antibody detection for diagnosis of acute malaria is usually not recommended, however, because the presence of antibodies can indicate past or recent infection. Enzyme-linked immunosorbent assays (ELISA) have been developed that use Plasmodium-derived antigens (Newmarket Laboratories, UK; Cellabs, Australia) or P. falciparum whole organism lysates (DiaMed) to detect immunoglobulins (IgG and/or IgM) in human serum or plasma. These assays are easier to perform, exhibit higher throughput and better sensitivity and specificity than IFA (Kitchen et al., 2004; Seed et al., 2005; Srivastava et al., 1991). Current commercial ELISA assays are insufficiently sensitive to detect antibodies directed against each of the four plasmodium species (She et al., 2007).
Antigens used to capture antibodies have included vaccine candidates. These antigens are attractive for diagnostic applications because these antigens are known to elicit antibody responses, and thus are likely to be useful to detecting antibodies produced by infected individuals that result from parasite infection. Examples of such antigens include circumsporozoite protein (CSP), apical membrane antigen 1 (AMA-1), merozoite surface protein (MSP) one and two, of both P. vivax and P. falciparum (Kitchen et al., 2004; Rodrigues et al., 2003). Other antigens of interest are MSP-2, -3, -4, -5, -8 -9, glutamate-rich protein, and serine repeat antigen (Girard et al., 2007).
Exported Protein-1 (EXP1; also known as QF116, antigen 5.1, and circumsporozoite related antigen (Meraldi et al., 2002)) has been studied in Plasmodium sp., although its ortholog in P. vivax has not been elucidated except by sequence gazing. In non-P. vivax species, the polypeptide is a vesicular protein that is thought to be important in intracellular transport of parasite proteins (Simmons et al., 1987). In P. falciparum, EXP1 is expressed as a 23 kD protein in the pre-erythrocytic and asexual blood stages of the parasite (Hope et al., 1984). An integral membrane protein, it is found in the membranes of parasitophorous vacuoles (endoplasmic and reticulum enshrouded vacuoles that protect intracellular parasites) and in vesicles within the host cell cytoplasm (Kara et al., 1990; Sherman, 1985; Tolle et al., 1993). Studies using an EXP1 murine homolog showed that the protein can induce protective T-cell immunity in mice against lethal challenges with P. yoelii (Charoenvit et al., 1999). Antibodies raised against P. falciparum EXP1 polypeptides have been successful in detecting malaria infections (Meraldi et al., 2002). Generally, the C-terminus is most antigenic in humans (Meraldi et al., 2002).
There have been reports of using P. vivax EXP1 sequences as tools to diagnose P. vivax infection (Kim et al., 2003; Son et al., 2001); however, these early efforts appear to have been based on incorrect sequences and the resulting diagnostics most likely detected P. falciparum EXP1 sequences. In both the Kim et al. (2003) and Son et al. (2001) reports, the authors used primer sequences apparently developed using the sequences disclosed by Simmons et al. (Simmons et al., 1987). Simmons et al. (1987) reported on P. falciparum EXP1 sequences, and noted that the sequence was highly conserved in five P. falciparum lines; however, Simmons et al. (1987) did not report on any EXP1 sequences from P. vivax. Kim et al. (2003) and Son et al. (2001) cite GenBank Accession No. X05074 as being from P. vivax; however, GenBank's entry indicates that this accession is part of P. falciparum. To circumvent this, Kim et al. (2003) and Son et al. (2001) used for a template blood from a vivax malaria patient, but data analysis suggests that the primers they used would not amplify P. vivax polynucleotide sequences because the last 3 nucleotides (3′) of the forward primer, and the last 6 nucleotides (3′) of the reverse primer do not anneal to the putative P. vivax EXP1 sequence as understood today.
Detection of antibodies in donated serum or plasma can be used to identify individual donors who have been exposed to malarial organisms and who may be recently infected and, therefore, potentially parasitemic. All four species of plasmodium that infect humans have been transmitted via blood transfusion, and though the incidence of post-transfusion malaria is low in the United States (Mungai et al., 2001), the availability of blood donors could be increased by implementation of plasmodium antibody screening assays such that only malaria-organism exposed individuals are deferred from blood donation rather than all donors who have traveled or lived in malaria endemic regions, as is the current practice. Such assays would theoretically detect antibodies against plasmodium species that infect humans and cause malaria (P. falciparum, P. vivax, P. ovale, and P. malariae). Commercial antibody ELISAs are currently in use (United Kingdom, Australia, France) or are being considered in other countries for the reinstatement of deferred donors (Elghouzzi et al., 2008; Kitchen et al., 2004; Seed et al., 2005). In these cases, donors are tested for antibodies to plasmodium derived antigens within several months of deferral.
A commercial assay (Pan Malaria Antibody CELISA) from Cellabs Pty. Ltd. (Brookvale, NSW, Australia) claims detection of antibodies to all four plasmodium species that cause malaria in humans and sensitivity of 94% versus immunofluoresence test (IFAT) (per package insert). Independent evaluation suggests the assay has poor sensitivity for falciparum and non-falciparum malaria antibody detection when compared to IFAT (Mertens et al., 1999). Independent evaluation of another assay from DiaMed AG (Switzerland) which utilizes a mixture of extracts of cultured P. falciparum and P. vivax recombinant protein (circumsporozoite protein), demonstrated poor sensitivity for detection of symptomatic individuals with microscopically confirmed P. vivax (18/24) but did detect antibodies in patients infected with P. ovale (2/2), or P. malariae (2/2) infection (Doderer et al., 2007). The malaria antibody assay manufactured by Newmarket Laboratories Ltd (Kentford, UK) claims detection of all four species of plasmodium responsible for human malaria though it contains only P. falciparum and P. vivax derived recombinant antigens. The package insert indicates sensitivity for P. ovale and P. malariae antibody detection of only 80% and 67%, respectively. Detection of antibodies among individuals infected with P. ovale or P. malariae may be due to past infection with either P. falciparum or P. vivax and hence reactivity is due to detection of persistent antibodies to these agents. Independent evaluation of the assay demonstrated detection of only 9/14 (64%) of patients with acute malaria due to P. ovale infection and 85% (15/18) of patients with P. vivax malaria (Kitchen et al., 2004). Hence, the claimed ability of these assays to detect human antibodies elicited by infection to P. falciparum as well as P. ovale, P. vivax and P. malariae is questionable. For those assays whose composition of solid phase antigen is known (e.g. Newmarket, DiaMed), the absence of P. ovale or P. malariae specific antigens suggests that detection of antibodies to these species may be due to antibody cross-reactivity which raises important questions about assay specificity as well as sensitivity, or the reactivity observed in P. ovale or P. malariae samples is due to the presence of P. falciparum or P. vivax antibodies from previous infections.
Thus, there is presently a significant need for reliable detection of plasmodium antibodies from P. vivax. 
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